1. Field of the Invention
This invention relates to a method and apparatus for cultivating anchorage and nonanchorage dependent cells.
2. Description of the Related Art
The cultivating of cells is desired for a variety of purposes such as for the production of hormones, enzymes, antibodies, vaccines and the like. Certain cell lines are anchorage dependent as is well known in the art. This means that the cells must adhere and grow as monolayers and multilayers anchored to a glass or plastic substrate.
Culture of anchorage dependent cells has conventionally been performed by attaching the cells to stacked petri dishes or by growing the cells in roller bottles. These approaches have the drawbacks of being unable to achieve high volumetric cell density or of having inadequate circulation necessary to the growth of the cells. In addition, cells tend to detach from their anchorage surface under conditions such as high or low serum concentrations, monolayer saturation or viscous sheer caused by stirring or perfusion of the culture medium.
Alternative methods have been suggested for increasing the surface area for attachment to increase proliferation of cells. Microcarrier systems have been described in which minute beads are formed of a specific gravity such that the beads float in culture fluid under gentle agitation. Once suspended, cells attach, spread and grow on the external surface of the microcarriers.
In particular, U.S. Pat. No. 5,114,855 describes using a microcarrier formed of microspheres. The microspheres have a diameter of less than about 60 .mu.m. Microcarrier systems have the disadvantage in that the necessity for suspension and mixing creates handling problems. Also, cells are subjected to mechanical stress which might result in cell rupture.
It is also known that certain types of cells can be grown in suspension. Suspension cell lines conventionally have been grown using a stirred-tank reactor. For certain applications, these suspension cells can be conveniently cross-linked to a solid matrix or encapsulated in gelatin, alginate, agarose, etc. to protect the cells from mechanical stress.
Conventional immurement methods have been used for facilitating the harvesting of cells in suspension culture. Examples of immurement techniques are: hollow fiber (Hopkinson, J. "Hollow Fiber Cell Culture Systems For Economical Cell-Product Manufacturing", Bio/Technology 3: 225-230 (1985)); and membrane reactors (Scheirer, W., "High Density Growth of Animal Cells Within Cell Retention Fermentors Equipped with Membranes", In: Spier, R. E. and Griffiths, J. B. (Eds.) Animal Cell Biotechnology, Vol. 3, pp. 263-281 (1988)). These techniques have the disadvantage that scale-up of these methodologies is difficult.
In addition, entrapment methods can be used for entrapping non-anchorage dependent cells within a matrix. A matrix used for entrapping cells is described in UK Patent No. GB 2178447. This patent describes a matrix of a nonwoven fabric having a pore diameter of from 10 .mu.m to 100 .mu.m for providing high internal volume with pores that are 1 to 20 times the volume of individual cells. A porous support sheet can be bonded to the matrix for providing dimensional stability and physical strength. The matrix sheets are used as liners for the bottom of petri dishes or are wound into a spiral and immersed in a container of medium. The matrix can also be used for growing anchorage dependent cells.
Packed bed reactors for the cultivation of anchorage dependent cells have been in use for many years. A packed bed bioreactor for growing either anchorage or nonanchorage dependent cells has also been described in U.S. Pat. No. 4,833,083. In this patent, a vessel contains a packed bed of a support or matrix material. Cells or cell components attach or are entrapped to the solid support material or matrix material of the packed bed. The solid support material is formed of diatomaceous earth, silica, alumina, ceramic beads, charcoal, polymeric beads or glass beads. Medium is pumped horizontally inwardly across the packed bed for radial flow of the medium through the packed bed.
Of possible general relevance are U.S. Pat. Nos. 5,081,036, 4,888,294, 5,100,799 and 5,019,512 related to impellers for generating flow of the medium within the cell culture. Other patents of possible general relevance to the invention are U.S. Pat. Nos. 4,087,327, 4,906,577, 5,079,161 and 5,126,269, 4,727,040 and 4,634,675 related to attachment surfaces and bioreactors.
A need exists for a device in which cells are protected against mechanical stress and the cells are provided with optimum levels of nutrients and oxygen. The highest possible cell densities or concentrations are needed to achieve economical and large quantity production of the cells of interest.